This project will investigate the biosynthesis and degradation of the beta-N-acetylhexosaminidase isoenzymes, Hexosaminidase A and Hexosaminidase B, and their interrelationships in rat kidney. The synthesis, intracellular transport, glycosidation and packaging of Hex A and Hex B will be investigated by a kinetic study of uptake of radioisotopically labeled sugars and leucine into the purified isoenzymes in subcellular fractions. Kidney slices will be incubated in vitro with (Cl4)- and (H3)-labeled precursors for various time periods, homogenized in cold 0.3 M sucrose and fractionated by a schema developed in this laboratory. A special rough microsomal fraction enriched in Hex and other hydrolases, and smooth microsomal, Golgi membrane and lysosomal fractions will be isolated. Fractions will be characterized ultrastructurally, biochemically and enzymatically. The Hex isoenzymes are extracted from fractions by a method designed to prevent autolytic degradation and isolated in a highly purified form by immunochemical precipitation, or by a combination of ion exchange chromatography and immunoprecipitation, for radioactivity measurements. Similar experiments will be performed in the intact animal after intravenous administration of labeled precursors. Catabolic turnover of the Hex isoenzymes in lysosomal fractions will be studied by measuring the decay rate of radioactivity of the isolated isoenzymes prelabeled in the peptide and carbohydrate portions of the molecule.